Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Sterols are essential structural and regulatory components of eukaryotic cellular membranes1,2. However, cholesterol over-accumulation is cytotoxic3 necessitating mechanisms to maintain this metabolite at appropriate levels. A pivotal component of this homeostasis is the esterification of free sterol by acyl-coenzyme A (CoA): cholesterol O-acyltransferase (ACAT)4,5. Indeed, the inhibition of ACAT in sterol-loaded cells induces cell death when extracellular sterol acceptors such as high density lipoproteins are absent6. Sterols are maintained at a high concentration in the plasma membrane (PM) relative to the endoplasmic reticulum (ER), where ACAT resides. Therefore, intracellular sterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, alter intracellular sterol transport, render cells nystatin sensitive, temperature sensitive, anaerobically inviable, and defective in sterol uptake. Human ARV1, a predicted ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.
Intracellular sterol esterification plays a critical role in maintaining cellular free sterol below toxic levels. Trafficking of sterol to ACAT is critical for this homeostasis. In order to identify genes involved in sterol trafficking, we screened for mutants of budding yeast (Saccharomyces cerevisiae) that require sterol esterification for viability. We have identified a novel yeast gene, ARV1 (termed xe2x80x98arvxe2x80x99 for xe2x80x98ARE2 Required for Viabilityxe2x80x99) that is essential when the otherwise dispensable yeast ACAT-homologous genes, ARE1 and ARE2, have been deleted from the genome. We have identified a human homolog, hARV1, which rescues yeast arv1 deletion mutants.
Arv1 mutant cells are temperature sensitive and nystatin sensitive, suggesting elevated free sterol. Arv1 cells are anaerobically inviable, suggesting a critical need for Arv1p when cells must obtain sterol from extracellular sources. Arv1 cells accumulate 50% of control levels of exogenously supplied C14 cholesterol, suggesting Arv1p may facilitate exogenous sterol transport. The Arv1p amino acid sequence predicts a novel transmembrane protein with high similarity to entries in the database from C. elegans and human. We identified full length cDNA clones corresponding to hARV1. hARV1 yeast expression plasmids were constructed and found to rescue arv1 deletion strains, including temperature sensitivity as well as anaerobic inviability. We propose that hARV1 in human cells might play a similar role to that of Arv1p in yeast, protecting them when intracellular sterol levels are too high or when ACAT activity is insufficient.
This invention provides an isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7.
This invention provides an isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7.
This invention provides an isolated nucleic acid which encodes yeast ACAT-Related Enzyme 2 Required for Viability protein (ARV1), wherein the encoded ARV1 protein has the amino acid sequence set forth in FIG. 5.
This invention provides a purified mammalian ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) having the amino acid sequence set forth in FIG. 7.
This invention provides a purified yeast ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) having the amino acid sequence set forth in FIG. 5.
This invention provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of complementary nucleotides present within a nucleic acid which encodes a mammalian ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1) having the amino acid sequence set forth in FIG. 7.
This invention provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of complementary nucleotides present within a nucleic acid which encodes a mammalian ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1) having the amino acid sequence set forth in FIG. 5.
This invention provides a method for identifying a chemical compound which is capable of inhibiting ACAT-Related Enzyme 2 Required for Viability protein (ARV1p) which comprises: (a) contacting an isolated ARV1p with the chemical compound under conditions permitting an effect to occur upon contact between the ARV1p and the chemical compound; and (b) determining whether the chemical compound inhibits the activity of the ARV1p so as to identify a chemical compound which is capable of inhibiting ARV1p.
This invention provides a method for identifying a chemical compound which is capable of stimulating ACAT-Related Enzyme 2 Required for Viability protein (ARV1p) which comprises: (a) contacting an isolated ARV1p with the chemical compound under conditions permitting an effect to occur upon contact between the ARV1p and the chemical compound; and (b) determining whether the chemical compound stimulates the activity of the ARV1p so as to identify a chemical compound which is capable of stimulating ARV1p.
This invention provides a pharmaceutical composition comprising ARV1 protein having the amino acid sequence set forth in FIG. 7 and a pharmaceutically acceptable carrier.
This invention provides a method of treating a subject who has a disease wherein cellular sterol transport or sterol uptake are defective comprising administering a pharmaceutical composition comprising ARV1 protein having the amino acid sequence set forth in FIG. 7 and a pharmaceutically acceptable carrier to the subject so as to restore the sterol transport, sterol uptake or sterol distribution.
This invention provides a method for determining whether elevated levels of intracellular sterol in absence of sterol esterification in a subject are due to ARV1 mutation which comprises: (a) obtaining a nucleic acid sample from the subject; (b) comparing the nucleic acid from the sample with the isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7; and (c) determining whether the nucleic acid from the sample of step (a) differs from the isolated nucleic acid encodes mammalian ARV1p, a difference in the nucleic acid from the isolated nucleic acid which encodes ARV1p indicating presence of a mutation in the nucleic acid encoding ARV1 from the sample.
This invention provides a method for determining whether elevated levels of intracellular sterol in absence of sterol esterification in a subject are due to ARV1 mutation which comprises: (a) obtaining nucleic acid from a sample from the subject; and (b) contacting the nucleic acid of step (a) with an isolated nucleic acid sequence encoding a mutant ARV1 under conditions permitting binding of any nucleic acid from the sample to the nucleic acid sequence encoding a mutant ARV1 so as to form a complex, formation of the complex indicating presence of a mutation in the nucleic acid encoding ARV1 from the sample.
This invention provides a method for treating a subject who has an imbalance in cell sterol levels due to a defect in sterol esterification which comprises introducing an isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7, into cells of the subject under conditions such that the nucleic acid expresses ARV1p and the cells obtain sterol from an extracellular source, so as to thereby treat the subject.
This invention provides a method for treating a subject who has an imbalance in sterol levels in cells due to a defect in sterol esterification which comprises administering ARV1p to the subject, such that the ARV1p transports to the cells extracellular sterol, so as to thereby treat the subject.
This invention provides a transgenic nonhuman mammal comprising an isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7.
This invention provides a transgenic nonhuman mammal comprising an isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7 which is mutated.
This invention provides an antibody directed to an epitope of a purified mammalian ARV1p having the amino acid sequence set forth in FIG. 7.
This invention provides a plant producing seeds whose lipid oils content is altered from the wild-type lipid oils content by a change in the expression of nucleic acid which encodes human ARV1 or plant ARV1.
This invention provides a plant seed whose lipid oils content is altered from the wild-type lipid oils content by a change in the expression of nucleic acid which encode human ARV1 or plant ARV1.
This invention provides a method of altering lipid oils content of a plant seed which comprises introducing into the nucleic acid of the plant seed an isolated nucleic acid encoding plant ARV1p , wherein the encoded plant ARV1p differs from the wild-type plant ARV1, such that the lipid oils content of the plant seed is altered.
This invention provides a pharmaceutical composition comprising lipid oils of a plant seed whose lipid oils content is altered from the wild-type lipid oils content of the plant seed by a change in the expression of nucleic acid which encode ARV1 and a pharmaceutically acceptable carrier.
This invention provides a method for identifying a chemical compound which has antifungal activity which comprises: (a) contacting a yeast cell comprising an isolated nucleic acid which encodes yeast ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein is a deletion mutant with the chemical compound under conditions permitting an effect to occur upon contact between the yeast cell and the chemical compound; and (b) determining whether the chemical compound inhibits yeast ARV1p so as to identify a chemical compound which has antifungal activity. The effect may be alteration of intracellular sterol transport, sensitivity to the compound, temperature sensitivity, anaerobic inviability, or a defect in sterol uptake.